Protective Effect of Bojungikki-Tang against Radiation-Induced Intestinal Injury in Mice: Experimental Verification and Compound-Target Prediction

Bojungikki-tang (BJIT) is a traditional herbal medicine used in Korea, Japan, and China to treat gastrointestinal disorders. In this study, we aimed to investigate whether BJIT has protective effects against radiation-induced intestinal injury and to predict the underlying therapeutic mechanisms and related pathways via network pharmacological analyses. BJIT was injected intraperitoneally (50 mg/kg body weight) to C3H/HeN mice at 36 and 12 h before exposure to partial abdominal irradiation (5 Gy and 13 Gy) to evaluate the apoptotic changes and the histological changes and variations in inflammatory cytokine mRNA levels in the jejunum, respectively. Through in silico network analysis, we predicted the mechanisms underlying BJIT-mediated regulation of radiation-induced intestinal injury. BJIT reduced the level of apoptosis in the jejunal crypts 12 h post 5-Gy irradiation. Histological assessment revealed intestinal morphological changes in irradiated mice 3.5 days post 13-Gy irradiation. Furthermore, BJIT decreased inflammatory cytokine levels following radiation exposure. Apoptosis, TNF, p53, VEGF, toll-like receptor, PPAR, PI3K-Akt, and MAPK signaling pathways, as well as inflammatory bowel disease (IBD), were found to be linked to the radioprotective effects of BJIT against intestinal injury. According to our results, BJIT exerted its potential protective effects by attenuating histopathological changes in jejunal crypts and suppressing inflammatory mediator levels. Therefore, BJIT is a potential therapeutic agent that can treat radiation-induced intestinal injury and its associated symptoms.


Introduction
Radiation therapy is crucial for the treatment of pelvic and abdominal malignancies, including carcinomas of the pancreas, cervix, ovary, prostate, uterus, and rectum [1,2]. Radiation therapy improves the prognoses of patients with malignancies since it directly eliminates the diagnosed malignancies or palliates symptoms associated with advanced, recurrent disease [1]. However, this therapy can lead to various complications, including radiation-induced intestinal injury [1][2][3]. Intestinal injury, a common complication with an incidence rate of 50-70% [4,5], leads to weight loss, diarrhea, intestinal strictures/fstulas, and even severe enteric septicemia, all of which can seriously afect the patient's quality of life [5,6].
Several efective compounds targeting radiation-induced intestinal injuries have been identifed [7][8][9][10]. Recently, natural products, especially herbal prescriptions in traditional medicine, have been assessed as potential radioprotective agents owing to their efcacy and low toxicity [11,12]. According to the theory of traditional medicine, ionizing radiation, which induces acute radiation injury (ARI), belongs to the "heat toxin" category. Heat toxin burns of Qi and Yin, which are integral substances of the human body and essential for the physiological activity of all organ systems [13]. In this respect, "clearing heat and removing toxin" and "tonifying Qi and nourishing Yin" are usually used together as the main principles for efective treatment [14].
Additionally, BJIT can be used for the treatment of radiotherapy-induced injuries. Many recent studies have indicated that the main herbal components in BJIT decoction have an antiradiation efect [11,20,21]. Moreover, a previous study that assessed intestinal crypt survival and apoptosis confrmed that BJIT treatment attenuates radiation-induced intestinal injury [11], indicating that BJIT might be a useful radioprotective agent. However, detailed mechanistic features remain unelucidated.
Te identifcation of complex molecular mechanisms is a major challenge with herbal formulas. Traditional herbal medicines are composed of multiple compounds, rendering the underlying mechanisms more complex than those of a single active compound [17]. However, the conventional experimental approach to determine mechanisms is timeconsuming, laborious, and expensive. Moreover, elucidating specifc interactions between compounds and their respective targets is difcult with the conventional approach [21]. Terefore, new methods and strategies are urgently needed to address this problem. Network pharmacology [22], a new strategy, can independently identify compoundtarget pathways related to a particular disease while providing a systematic and holistic view [23].
In this study, we examined the protective efect of BJIT against intestinal injury in a murine model exposed to radiation. Ten, to holistically evaluate the regulatory mechanisms of BJIT, we performed a pharmacological network analysis of BJIT to predict the potential active compounds and radiation-induced intestinal injury-related target genes via in silico network analysis. . Animals were maintained in a room at 23 ± 2°C with a relative humidity of 50 ± 5%, artifcial lighting from 08:00-20:00, and 13-18 air changes per hour. Mice were fed a standard animal diet. Experiments were performed one week after quarantine and acclimatization. All the animal procedures were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Oriental Medicine (KIOM 20-032) and were performed in compliance with the National Institutes of Health Guidelines for the care and use of laboratory animals and the Korean national laws for animal welfare.

Preparation of Sample Solutions.
A decoction of BJIT was prepared in our laboratory from a mixture of chopped crude herbs (37.5 g of A. gigas, 112.5 g of A. membranaceus, 75 g of A. japonica, 22.5 g of B. falcatum, 22.5 g of C. heracleifolia, 37.5 g of C. unshiu, 75 g of P. ginseng, and 75 g of G. uralensis), which were extracted in 5 L of distilled water at 100°C for 2 h. Te solution was evaporated to dryness and freeze-dried (extract: 119.2 g; yield: 26.05%). Te lyophilized BJIT extract was dissolved in distilled water and mixed.

Irradiation Exposure and Experimental Groups.
Each mouse was anesthetized with 85 mg/kg of alfaxalone (Alfaxan ® ; Careside, Republic of Korea) and 10 mg/kg of xylazine (Rompun ® , Bayer Korea, Republic of Korea) and restrained on a tray. Mice were exposed to abdominal radiation using 6 MV high-energy photon rays (ELEKTA, Stockholm, Sweden) at a dose of 3.8 Gy/min. Abdominal irradiation at doses of 5 Gy and 13 Gy was used to evaluate apoptotic changes (Experiment 1) and histological changes (Experiment 2) in the jejunum, respectively. Shamirradiated mice were treated the same way as radiated animals but without radiation. Te experimental timeline is summarized in Figure 2.
In the frst set of experiments, to evaluate the efect of BJIT on apoptotic changes, mice were divided into four groups that received the following treatment regimens: (Exp.   In experiment 1, the mice were pretreated with intraperitoneal injections of vehicle or BJIT decoction at 36 h and 12 h before irradiation. Ten, the mice received partial abdominal irradiation at 0 (Sham) or 5 Gy and were euthanized after 12 h In experiment 2, the mice were pretreated with intraperitoneal injections of vehicle or BJIT decoction at 36 and 12 h before radiation exposure. Ten, the mice were irradiated with 0 or 13 Gy and were euthanized for tissue sampling at 3.5 days postirradiation. Black circles indicate the times of tissue collection from the sham-irradiated (0 Gy) controls and irradiated (5 or 13 Gy) test animals.

Histological Changes in the Jejunal Crypts.
For histological analysis, two slices from each mouce's jejunum were sectioned from four diferent spots. Hematoxylin and eosin (H&E) was used to stain intestinal slices in order to examine the morphology. Ten the jejunal cross-sections were counted for the regenerating crypts and villi. All samples were sectioned and reoriented in successive slices to determine which ones had the longest villi in order to analyze the morphological changes. Tis method was chosen because it produced data that were more consistent than that produced by normal methods, which only measured the ten longest villi in a single slice per sample [8,9]. Te lengths of the ten longest villi and the heights of the basal lamina of ten small intestinal sections from each animal were measured. Using a polyclonal rabbit anti-KI-67 antibody (Acris Antibodies GmbH, Hiddenhausen, Germany; diluted 1 : 500), the proliferation was examined using immunohistochemistry to measure cell proliferation in jejunum samples. Avidin-biotin peroxidase (Elite Kit, Vector Laboratories, Burlingame, CA, USA) was used to identify the attached antibodies, and a diaminobenzidine substrate kit (Vector) was used to assess the peroxidase activity. In each experiment, the primary antibody was left out of a few test sections as a negative control. A digital camera mounted on a microscope was used to capture images of intestinal sections (Leica DM IRBE, Leica MicroSystems GmbH, Wetzlar, Germany). Utilizing software for image processing, quantifcation was performed (Leica QWin, Leica Microsystems, Wetzlar, Germany).

Determination of mRNA Levels by Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR).
Total RNA was extracted from small intestine tissues using an ISOGEN kit (Nippon Gene, Tokyo, Japan). Real-timeqRT-PCR analyses were performed as previously described [25]. Te expression levels of mRNAs for Bax, Bcl2, iNos (Nos2), and Cox-2 (Ptgs2) were quantifed, normalized to the expression level of β-actin (Actb) mRNA, and expressed relative to the corresponding mean value for the small intestine tissue of sham-irradiated control mice. Te sequences of the PCR primers and TaqMan probes are listed in Table 1 5.0, accessed on August 24, 2020) with "Homo sapiens" selected as the organism [26]. Tis database provides a platform for exploring known interactions between small molecules, proteins, and organism-basedprotein-protein interactions [27]. Active compound-protein interactions with an interaction score ≥0.400 (medium confdence) were selected [28]. Gene information, including gene IDs and names, was verifed in the UniProt database (https://www. uniprot.org/, accessed on September 15, 2020) by limiting to "Homo sapiens" as the species.

Potential Target Genes and Protein-Protein
Interaction. Te public database GeneCards: Te Human Gene Database (https://www.genecards.org/, version 5.0, accessed on October 14, 2020) was searched for information on potential target genes, limiting to those from qRT-PCR, quantitative reverse transcription polymerase chain reaction; FWD, forward; RVS, reverse.
"Homo sapiens." Te aforementioned target genes were matched to intestinal injury-related genes, and overlapping genes were consolidated as potential target genes. Using a high confdence score (≥0.700), a protein-protein interaction (PPI) network for "Homo sapiens" was created with the STITCH database (https://stitch.embl.de/, version 5.0) [29].

Statistical Analysis.
Te results are reported as the mean ± standard error of the mean (SEM) and were analyzed using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post hoc test for multiple comparisons. In all cases, a p value <0.05 was considered signifcant.

Protective Efects of BJIT against Intestinal Apoptotic
Changes in Irradiated Mice. In the jejunal crypts, apoptosis was easily recognized in apoptotic bodies by TUNEL staining of the tissue postradiation. Most apoptotic cells were observed in the putative stem cell zone located at the base of the jejunal crypts (Figure 3(a)). In the irradiated group, a marked increase in the number of apoptotic nuclei was observed at the base of the jejunal crypts (sham: 0.09 ± 0.02, 5 Gy: 3.10 ± 0.19, p < 0.001; Figure 3(b)). BJIT treatment signifcantly reduced this parameter (BJIT + 5 Gy: 2.31 ± 0.17, p < 0.05; Figure 3(b)). We analyzed the expression levels of proapoptotic and antiapoptotic mRNAs, namely, Bax and Bcl-2, respectively, in the small intestine 12 h postirradiation. As shown in Figure 3(c), the irradiation-mediated apoptosis was characterized by a marked increase in the Bax mRNA level (sham: 1.22 ± 0.28, 5 Gy: 2.68 ± 0.59, p < 0.05). BJIT treatment did not signifcantly reduce this parameter (BJIT + 5 Gy: 1.88 ± 0.44, p � 0.30; Figure 3(c)). As shown in Figure 3(d), the level of Bcl-2 mRNA remained unchanged in all groups, indicating that Bcl-2 mRNA was constitutively expressed in the jejunal tissue and was not signifcantly altered after 5-Gy irradiation (sham: 1.14 ± 0.35, 5 Gy: 0.72 ± 0.15, p � 0.29). Although no statistically signifcant diference was detected, treatment of BJIT apparently

Protective Efect of BJITas Demonstrated by Infammatory Cytokine Levels in Intestinal Samples from Irradiated Mice.
We studied the mRNA expression levels of infammatory cytokines (i.e., Cox-2 and iNos) in the small intestine 3.5 days postirradiation. As shown in Figure 6 Figure 6(a)). As shown in Figure 6(b), the iNos mRNA levels in the 13 Gyirradiated mice signifcantly increased postirradiation (sham: 1.12 ± 0.21, 13 Gy: 7.42 ± 1.23, p < 0.001). However, BJIT treatment did not signifcantly reduce this parameter (BJIT + 13 Gy: 5.31 ± 1.06, p � 0.08; Figure 6(b)). Although no statistically signifcant diference was detected, BJIT treatment did attenuate the increase in Cox-2 and iNos mRNA levels.  Evidence-Based Complementary and Alternative Medicine 7 TCMSP guidelines [14,15]. Although ten active compounds, namely, astragaloside IV; ferulic acid (cis and transforms); ginsenosides Rb1, Re, and Rg1; glycyrrhizin; hesperidin; isoferulic acid; and saikosaponin A, did not meet the ADME criteria, they were included because they are major active compounds according to the Korean and Chinese Pharmacopoeia. A total of 173 active compounds were selected through ADME screening and Pharmacopoeia guidelines (Supplementary Table 2).

Selection of Potential Target Genes.
Forty-four active compounds were linked to 772 target genes in the STITCH database with scores ≥0.400 (medium confdence, Supplementary Table 3) [28]. Next, these genes were matched with intestinal injury-related genes (n � 8262) in the GeneCards database (Supplementary Table 4), and only genes with scores ≥0.700 were selected. Finally, seven herbs, 505 target genes, and 37 active compounds were selected, and this network consisting of herbs-compounds-genes (H-C-G) was composed of 549 nodes and 808 edges (Figure 7). Ten, the target genes were matched with intestinal injury-related genes in the GeneCards database. In total, 406 potential target genes overlapped with disease-associated genes. Tese potential target genes helped produce a PPI network with the STITCH database, and their topology was analyzed in the Cytoscape program. A topological module represents a locally dense neighborhood in a network, such that nodes have a higher tendency to link to the nodes within the same local neighborhood than to the nodes outside of it [31]. Te PPI network consisted of 505 potential target genes, and TP53, AKT1, PPARA, JUN, MAPK14, STAT3, BCL2, PPARG, TNF, EGFR, SP1, VEGFA, MMP9, and MAPK3 were determined to be core potential genes (Figure 8). Tese genes had high-degree edge counts and were closely related to intestinal injury.

Discussion
Bojungikki-tang is a popular traditional medicine in Korea, China, and Japan [14,15]. According to the theory of traditional herbal medicine, each of the eight herbs in BJIT has medicinal efects. Briefy, Astragali Radix, Ginseng Radix, Atractylodis Rhizoma Alba, and Glycyrrhizae Radix et Rhizoma can reinforce Qi. Citri Unshius Pericarpium can regulate Qi, and Angelica Gigantis Radix can "tonify blood." Cimicifugae Rhizoma and Bupleuri Radix can elevate Yang-Qi. Te complex of eight herbs can "tonify the middle" and augment Qi ( Figure 10). Furthermore, a defciency in middle Qi mainly results in gastrointestinal symptoms, including dyspepsia, inappetence, diarrhea, and epigastric discomfort. Tus, clinically, BJIT is generally used to treat various gastrointestinal disorders, including radiation therapy-induced intestinal injury [17].
In a previous study, we demonstrated that BJIT could attenuate radiation-induced intestinal injury [11]. To confrm the efects of this herbal prescription, we attempted to verify the efcacy of BJIT in protecting against radiationinduced intestinal injury and further explored the potential molecular mechanisms of BJIT components through a systematic approach using network pharmacology.
Te pathogenesis of radiation-induced intestinal injury is multifactorial and mainly related to cell apoptosis in the crypt epithelium and infammatory processes [5,32]. Apoptosis is one of the most important outcomes of irradiation-mediated intestinal damage. Upon radiation exposure, an imbalance between apoptotic and antiapoptotic factors occurs within the cells [33][34][35]. Te decreased level of Bcl-2, an antiapoptotic factor, and simultaneous accumulation of Bax, a proapoptotic factor, is associated with an augmentation of the apoptotic response [36]. In the PCR analysis of the present study, 5-Gy irradiation resulted in increased Bax expression and decreased Bcl-2 expression levels compared to sham mice. In this context, although BJIT elevated Bcl-2 levels and suppressed the increased Bax expression, no statistically signifcant diferences were noted. Te TUNEL assay revealed that BJIT treatment signifcantly mitigated the number of apoptotic nuclei within the jejunal crypts. Overall, these results show that BJIT administration attenuated the irradiation-induced apoptosis in the intestinal crypts.
Radiation exposure generally suppresses cell proliferation in the crypts, delaying the development of intestinal damage [31,37]. Tis loss of proliferative function could exacerbate mucosal infammation and dysfunction by augmenting intestinal permeability to luminal antigens and bacteria [3]. Hence, the number of surviving crypts and villus length can be used as biodosimetry markers to investigate the adverse efects of radiation [10,38]. In this  study, the villus length of the irradiated mice was signifcantly shorter than that of the sham mice. Additionally, the irradiated mice showed signifcantly increased mucosal depth. Conversely, BJIT treatment ameliorated these radiation-induced histopathological changes. Furthermore, we investigated the changes in the expression level of Ki-67, a proliferation marker, in the jejunum via immunohistochemical staining. Irradiation suppressed the number of Ki-67-positive crypts, refecting a decrease in surviving crypts. Te BJIT-treated mice showed a greater number of Ki-67positive crypts compared to the irradiated group. In this study, BJIT treatment resulted in larger crypt sizes following irradiation, which is one of the indications of crypt regeneration [39,40].
Tere is growing evidence supporting the hypothesis that infammation is involved in the development and pathogenesis of radiation-induced injury in normal tissue [41]. Radiation stimulates the translocation of nuclear factorkappa B (NF-κB) to the nucleus, increasing the expression of proinfammatory mediators, including iNOS [42,43]. iNOS levels in the intestines of rats were reportedly elevated as early as 2 h after radiation treatment [44]. Te elevated iNOS levels result in COX-2 overexpression, which produces prostaglandins via the metabolism of arachidonic acid [42,43]. In this study, radiation exposure increased Cox-2 and iNos mRNA levels in the intestine. Although BJIT administration attenuated the upregulation of Cox-2 and iNos mRNA levels, the diferences were not statistically signifcant.
Using network analysis, 1,319 active compounds were extracted from public databases, and 34 active compounds that passed ADME screening were linked to intestinal injury-related genes. BJIT consists of eight herbs, of which Atractylodis Rhizoma Alba was excluded from the network because its active compounds did not exhibit interactions with genes linked to intestinal injury, although it has been used to treat digestive disorders [45]. Beta-sitosterol and stigmasterol in BJIT are classifed as plant sterols, and previous studies have reported that plant sterols reduce systemic infammatory responses [46]. In this context, many favonoids can infuence chronic infammatory disease at the cellular level and modulate the responses of protein pathways [47]. Seventeen of the 37 compounds in BJIT are categorized as favonoids in this network. Notably, hesperidin is a favanone glycoside mainly found in citrus fruits, which reportedly exhibit anti-infammatory, antimicrobial, anticarcinogenic, and antioxidant efects and is efective in reducing the intensity of small intestine damage [48]. Additionally, favonoid-rich fractions were able to modulate the NF-κB signaling pathway in a previous study [49]. Tus, these favonoids in the BJITnetwork might play a vital role in reducing infammation.
Te core node components linked to intestinal injuryrelated genes included TP53, AKT1, PPARA, JUN, MAPK14, STAT3, BCL2, PPARG, TNF, EGFR, SP1, VEGFA, MMP9, and MAPK3. In the DAVID and KEGG network pharmacology analysis, intestinal injury-related genes were associated with nine pathways. Te apoptosis and p53 pathways are associated with radiation-induced gastrointestinal disturbances, particularly the damage to the small intestine. Te antiapoptotic efect has been shown to play one of the most important roles in radioprotection [49][50][51]. Among the nine pathways, the PI3K-Akt and NF-κB-mediated signaling pathways were closely correlated with the pathogenesis of gastric disease and intestinal mucosal injury [52]. Toll-like receptor (TLR) signaling plays important roles in maintaining intestinal epithelial homeostasis [53]. However, harmful factors in the intestinal tract, such as infammatory cytokines, activate TLR signaling. TLR activates downstream signaling pathways involving Myd88 to induce nuclear translocation of NF-κB, thereby increasing proinfammatory cytokine production [54]. Signaling by TLRs on intestinal epithelial cells is critical for intestinal injury [55]. TLRs and their ligands provide novel strategies for radiation protection during nuclear accidents as well as for the protection of normal tissues during cancer radiotherapy [56]. Targeting TLRs may represent a novel therapeutic approach in cancer therapy-induced intestinal mucositis. Peroxisome proliferator-activated receptor c (PPARc) is a nuclear receptor highly expressed in the intestines and plays a key role in infammation. Further studies should explore the efects of abdominal irradiation on PPARs, their roles and functions in irradiation toxicity, and the possibility of using their ligands for radioprotection [57].

Conclusions
In summary, BJIT exerts signifcant protective efects against radiation-induced intestinal injury in a murine model by alleviating the extent of histopathological changes in jejunal crypts and suppressing the levels of infammatory mediators. Moreover, through subsequent network analysis, we identifed TP53, AKT1, PPARA, JUN, MAPK14, STAT3, BCL2, PPARG, TNF, EGFR, SP1, VEGFA, MMP9, and MAPK3 as potential target genes playing pivotal roles in various signaling pathways related to the radioprotective efects of BJIT.

Data Availability
Te data that support the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
All procedures were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Oriental Medicine (KIOM 20-032) and were performed in compliance with the National Institutes of Health Guidelines for the care and use of laboratory animals and the Korean national laws for animal welfare.

Conflicts of Interest
Te authors declare that they have no conficts of interest.
Soong-In Lee, Hyun-Yong Kim, Jung Min Lee, Changjong Moon, In Sik Shin, Sung-Wook Chae, Jihye Lee, Yun-Soo Seo, and Joong-Sun Kim performed investigation. A Yeong Lee provided the resources. Sohi Kang and A Yeong Lee wrote the original draft. Jihye Lee, Yun-Soo Seo, and Joong-Sun Kim reviewed and edited the manuscript. All authors have read and approved the fnal version of the manuscript.